The present invention relates to a tissue culture medium composition useful for induction and proliferation of calli from various explants of mature trees of Taxus baccata ssp. Wallichiana Zucc. Pilg., commonly known as the Himalayan Yew. The invention also relates to a process for production of Taxus plants using the said medium and production of taxane class of anti-cancer compounds in these callus cultures in higher yields.
Since the discovery of novel anticancer properties of taxol, a highly derivatized diterpenoid first isolated from Taxus brevifolia (Wani et al., J. Am. Chem. Soc. 93: 2325-2327, 1971), considerable interest has been generated about the plants of this genus. All the plant parts from all the species of Taxus are reported to contain taxol and a number of other related taxanes with diverse clinical activities. 10-deacetylbaccatin-III (10-DAB) is one such taxol precursor, which is useful in the semi-synthesis of taxol. The yields of taxol vary from 0.0040% to 0.1% on dry weight basis (Vidensek et al., J. Nat. Prod. 53: 1609-1610, 1990; Witherup et al., J. Nat. Prod. 53: 1249-1255, 1990). The yews have become endangered in their natural habitats because of their over-exploitation and inherent slow growth. Use of plant tissue and cell culture technologies could potentially provide sufficient quantities of taxol and other related, clinically active taxanes for human use, without resorting to the cutting down of the yew trees for extraction of the taxanes.
A number of reports have either been published or patented as listed in the review by Jaziri et al. (Plant Cell Tissue Organ Cult. 46: 59-75, 1996 and references cited therein), related to taxol and/or taxane production in vitro from callus cultures derived from either Pacific or European or Japanese or Korean Yews. Taxus baccata ssp. Wallichiana Zucc. Pilg., Himalayan Yew is an endangered gymnosperm growing at altitudes of above 3000 m. Not much work has been done on these plants. There are no reports pertaining to taxol and/or 10-DAB production in tissue cultures of the Himalayan Yew except for the paper on the production of 2-deacetoxytaxinine and 2-deacetoxyaustrospicatine from callus cultures of Taxus wallichiana (Banerjee et al., Planta Med. 62: 333-335, 1996). The composition of the basic medium published earlier by Gamborg et al. (1968) is as follows:
Several patents disclose media and methods for production of taxol from Taxus sp. ex-plants.
The medium compositions described in the prior art are not useful for formation and proliferation of callus lines of Himalayan Yew. Hence, the applicants have developed a novel medium suitable for development of callus lines of Taxus sp. such cultures are capable of producing high amount of Taxol.
The main object of the present invention is to provide a culture medium for induction and proliferation of callus lines from mature explants of Taxus baccata ssp. Wallichiana Zucc. Pilg., Himalayan Yew.
Another object of the invention is to provide a method for production of Taxus plants using the culture medium of the invention.
Another object is to provide a process for the production of taxol and 10-DAB from callus cultures of Taxus baccata ssp. Wallichiana Zucc. Pilg.; which is a native tree of India.
Another objective is to report an optimized culture medium composition for induction and proliferation of callus cultures from a number of explants like stems, needles, embryos and endosperms from mature trees of Taxus baccata ssp. Wallichiana Zucc. Pilg., Himalayan Yew.
Another objective of the present investigation is to provide a composition of the culture medium including appropriate concentrations of macronutrients, micronutrients, vitamins, plant growth regulators and antioxidants to obtain fast growing callus cultures from selected explants of Taxus baccata ssp. Wallichiana Zucc. Pilg., Himalayan Yew.
Yet another objective is to provide a composition capable of sustaining the growth of callus cultures and inducing high production of taxane class of molecules under a given set of incubation conditions.